The hepatitis C virus (HCV) was discovered and identified as the causative virus of non-A, non-B hepatitis by Choo et al. in 1989 (Non-Patent Document 1). HCV infection causes chronic hepatitis, and the chronic hepatitis progresses to cirrhosis with persistent HCV infection, and then to liver cancer. It is said that approximately 170,000,000 patients are infected with HCV in the whole world, and approximately 2,000,000 patients are infected therewith in Japan. HCV is mainly transmitted through blood. Although the number of patients newly infected with HCV was sharply reduced since screening of blood for transfusion was made possible, it is considered that a large number of virus carriers still exists.
At present, treatment of chronic hepatitis C is mainly carried out via administration of pegylated interferon or combination therapy with pegylated interferon and the anti-virus agent ribavirin. Up to the present, HCV has been classified into 6 different genotypes. Infection with HCV genotypes 1b and 2a are major cases in Japan. In particular, viruses of HCV of genotype 1b cannot be completely removed from the body by the administration of interferon in combination with ribavirin, and the therapeutic effects are not satisfactory (Non-Patent Documents 2 and 3). Accordingly, development of novel anti-viral agents or vaccines aimed at the prevention of development of hepatitis C or the elimination of HCV viruses has been awaited.
Virus vaccines are classified based on antigens; that is, component vaccines using viral proteins as antigens; vaccines using virus particles as antigens; and DNA vaccines using viral protein-encoding genes. Vaccines using virus particles as antigens are classified as attenuated live vaccines or inactivated vaccines. When vaccines using virus particles as antigens are produced, a system for producing highly purified virus particles is necessary, and such system requires a culture system for producing large quantities of virus particles.
The hepatitis C virus (HCV) comprises a plus single-stranded RNA genome of approximately 9.6 kb. The HCV single-stranded RNA genome encodes a single polyprotein (i.e., a polyprotein precursor) containing 10 types of proteins (i.e., Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins). A polyprotein precursor translated from the HCV RNA genome is cleaved into individual proteins, so as to serve as viral proteins.
A replicon system that allows autonomous replication of HCV RNA in a cell culture system has been developed and employed in many studies regarding HCV. A typical subgenomic replicon is prepared by substituting a structural protein region of HCV genome with a marker gene, such as a drug resistance gene, and inserting IRES from encephalomyocarditis virus (EMCV) into a site downstream thereof. Replication of HCV RNA is observed in cultured cells into which the subgenomic replicon RNA has been introduced (Patent Document 1). Studies on the replication of HCV subgenomic replicon show that genetic mutations of the HCV genome may exhibit the effect to enhance the replication efficiency of replicon, and such genetic mutations are referred to as adaptive mutations (Patent Document 1).
NK5.1 strain (Con1/NK5.1), which is a variant of the subgenomic replicon pFK-I389neo/NS3-39/wt (Con1/wt) derived from the Con1 strain of genotype 1b and has an adaptive mutation in the NS3-NS5A region, is reported to have proliferative capacity approximately 10 times higher than that of the wild-type Con1/wt strain (Non-Patent Document 4). Meanwhile, the literature describing the results of sequence analysis of replicons in replicon-replicating cells having subgenomic replicons derived from the HCV JFH1 strain of genotype 2a isolated from a patient with fulminant hepatitis (Non-Patent Document 5) discloses that several mutations were observed in the HCV genome-derived regions in 5 out of 6 resulting clones, but no common mutations were observed among them. In addition, the literature discloses that a nucleotide mutation in the other one clone would not cause amino acid mutation. This indicates that the JFH1 strain is capable of proliferating in Huh7 cells without adaptive mutations.
Regarding HCV production in a cell culture system, Wakita et al. showed that infectious HCV particles were successfully produced via introduction of the full-length HCV genomic replicon derived from the JFH1 strain into Huh7 cells (Patent Document 2 and Non-Patent Document 6). Also, Kaul et al. reported that the mutations in the NS5A protein of the JFH1 strain resulted in the production of viruses in amounts approximately 10 times higher than that of the wild-type JFH1 strain (Non-Patent Document 7).
It is reported that the capacity of the JFH1 strain for virus particle production in a cell culture system is 4.6×104 FFU/ml (Non-Patent Document 8), which is much lower than the capacity of influenza virus for virus particle production in a cell culture system, i.e., about 4×109 PFU/ml (Non-Patent Document 9). Production of vaccines using HCV particles as antigens requires the development of HCV strains with a higher capacity for virus particle production.